Journal of Clinical Virology 61 (2014) 189–195

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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv

Review

Medical and molecular perspectives into a forgotten epidemic: Encephalitis lethargica, viruses, and high-throughput sequencing Dennis Tappe a,∗,1 , David E. Alquezar-Planas b,∗∗,1 a b

Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Denmark

a r t i c l e

i n f o

Article history: Received 24 April 2014 Received in revised form 21 July 2014 Accepted 22 July 2014 Keywords: Encephalitis lethargica Postencephalitic parkinsonism Brain samples Virus Paleo-virology High-throughput sequencing

a b s t r a c t The emergence of encephalitis lethargica (EL), an acute-onset polioencephalitis of unknown etiology as an epidemic in the years 1917–1925 is still unexplainable today. Questioned by the first descriptor of EL himself, Constantin von Economo, there has been much debate shrouding a possible role of the “Spanish” H1N1 influenza A pandemic virus in the development of EL. Previous molecular studies employing conventional PCR for the detection of influenza A virus RNA in archived human brain samples from patients who died of acute EL were negative. However, the clinical and laboratory characteristics of EL and its epidemiology are consistent with an infectious disease, and recently a possible enterovirus cause was investigated. With the rapid development of high-throughput sequencing, new information about a possible viral etiology can be obtained if sufficient specimens for analysis were still available today. Here, we discuss the implications of these technologies for the investigation of a possible infectious cause of EL from archived material, as well as a prospectus for future work for acquiring viral nucleic acids from these sources. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Is EL an infectious disease? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuropathology findings support a viral etiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The investigations on influenza A virus as a possible cause and the role of archived material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Further viral candidates and the hypothesis of pathogen-induced autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paleovirology and high-throughput sequencing pipelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ∗ Corresponding author at: Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany. Tel.: +49 40 42818252; fax: +49 40 42818211. ∗∗ Corresponding author at: Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5–7, DK-1350 Copenhagen, Denmark. Tel.: +45 353 21236. E-mail addresses: [email protected] (D. Tappe), [email protected] (D.E. Alquezar-Planas). 1 Both the authors contributed equally to this work. http://dx.doi.org/10.1016/j.jcv.2014.07.013 1386-6532/© 2014 Elsevier B.V. All rights reserved.

The emergence of encephalitis lethargica (EL, “von Economo’s disease”), an acute-onset epidemic polioencephalitis of unknown etiology, remains unexplainable today. In recent years, EL has experienced a renewed interest in its etiology due to the application of molecular diagnostic methods on archival brain material. The disease was reported around the globe from 1917 to 1925, and to a lesser extent until 1940, with the majority of cases

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Fig. 1. Constantin Baron von Economo (1876–1931), Austrian psychiatrist and neuroscientist. Besides the description of encephalitis lethargica, von Economo extensively published work on many aspects of neuroscience, including the famous cytoarchitectonic descriptions of cortical areas.

described in England, the United States, Canada, Germany, and the Soviet Union [1]. The number of reports peaked in 1921 [1] with more than 1 million cases and half a million deaths [2] up to the 1920s. The first denominator of this new disease was Constantin Baron von Economo (1876–1931; Fig. 1), an Austrian physician of Greek origin. He presented his clinical and pathological investigations in 1917 (Fig. 2). Nearly simultaneously, René Cruchet reported a similar syndrome in France, without recognizing it as a distinct disorder [3,4]. Von Economo however had grouped the complex neuropsychiatric symptoms of the acute illness in three predominant clinical syndromes: somnolentophthalmoplegic, hyperkinetic, and amyostatic-akinetic [5,6]. Of those who fell ill, approximately one third died acutely, one third recovered and the remaining third developed sequelae, most often postencephalitic parkinsonism (PEP) [1,6]. PEP developed either immediately after the acute phase of EL or weeks to years later, with oculogyric crises as a characteristic symptom. Although sporadic cases resembling EL are occasionally described today, the disease has disappeared in its epidemic form [7]. An infectious cause, presumably of viral origin as direct cause of EL and/or a pathogen-driven autoimmune reaction following such an infection as cause of PEP, remains very plausible today based on epidemiological, clinical, laboratory, and pathology data. Because of the lack of techniques for proper virus isolation and molecular characterization at the time of the epidemic, EL as a clinical entity rests on historical descriptions and contemporary laboratory data [8]. The rapid current development of high-throughput sequencing (HTS) provides an opportune time for retrospective investigations of historical diseases and can be used to gather new information about the pathogen presumably involved in the etiology of EL. In this article, we review work on candidate microbes and discuss the implications of HTS for possibly resolving the question of which pathogen caused EL. 2. Is EL an infectious disease? The comparatively rapid emerging epidemic spread onset in the winter months in the northern hemisphere (December–March), and prodromal symptoms make an infectious cause of EL highly likely. This was already debated at the time of the EL epidemic in the light of the roughly contemporary and often lethal 1918/1919 ‘Spanish’ (H1N1) influenza. The assumption that EL was viral in origin was already prevalent in 1921 and has continued to be so to the present [2,9,10]. Prodromal symptoms of acute EL included

in some patients, but by far not all, pharyngitis, respiratory symptoms, parotitis, and fever [6]. The patient’s body temperature did not follow any observable patterns and was described with either continuous or intermittent fever, and even subfebrile [6]. Some had bradycardia, many were anorectic and lost weight. The occurrence of scarlet fever-like exanthemas and purpura-like lesions was reported [6]. Most certainly however, EL and PEP were overdiagnosed at that time [11], and not all symptoms may have been due to EL. The incubation period was not well defined, ranging from one day to two months [6]. The vast majority of patients died within two weeks of initial symptoms. Laboratory investigations showed peripheral blood leukocytosis, a relative lymphopenia, and monocytosis. Cerebrospinal fluid examinations revealed a lymphatic pleocytosis, with elevations of glucose and protein levels [6]. EL transmission is perplexing, as the evidence for person-toperson transmission is weak [12]. The spread of the disease was sporadic, without obvious relation to economic class, geography, or age group [8]. Outbreaks, such as in a girl’s school home, have been described [13]. However, these were rare and researchers believed healthy carriers were responsible for the spread of EL [12]. Von Economo speculated about a droplet transmission during the prodromal stage [6]. Although some authors considered toxins and chemical agents as the cause of EL, laboratory and neuropathological findings were consistent with an infectious, and most likely viral, cause. 3. Neuropathology findings support a viral etiology Neuropathology of acute EL cases from the epidemic showed widespread diffuse hemorrhagic and inflammatory lesions [14]. Von Economo described the disseminated EL lesions as an acute polioencephalitis predominantly affecting the midbrain in contrast to the rare hemorrhagic edematous leukoencephalitis seen in influenza [6]. The pons, medulla, thalamus, and basal ganglia were also affected. Typical, pathology in these regions consisted of perivascular infiltration with lymphocytes and plasma cells, and congestion [1]. Cortical EL lesions were typically confined to one lobe. There was nerve cell death, neuronophagy, and astrocytosis. In general, there was a poor relationship between clinical symptoms and neuropathological findings, which is otherwise seen in degenerative brain diseases [15]. However, the astrogliosis found in EL supported a viral etiology [16], and experimental animal studies with filtered and unfiltered brain suspensions and homogenates from patients who had died of acute EL led in some cases to similar pathology in animals. The effects on the infected monkeys, guinea pigs and rabbits were variable, however, it was later largely accepted (but not clearly proven) that EL is transmissible and would be due a filterable virus [9,17]. Notably however, by today’s standards some of these experiments were poorly controlled and misinterpreted [8]. 4. The investigations on influenza A virus as a possible cause and the role of archived material In recent years, there has been much continued debate regarding influenza as a potential cause of EL. However, already in his early publications von Economo had refuted the association [6], as the timeline of both diseases were inconsistent [6,9]. The reported cases of EL by von Economo (1917) predate the beginning of the “Spanish” influenza (1918) by at least one year [6]. A causal relationship has never been documented [18]. Whereas the 1918 influenza pandemic spread from North America to Europe, EL has spread from Europe to North America [19]. Moreover, while influenza is very contagious, EL was apparently not. At the time of both

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Fig. 2. May 1917 issue of the Wiener klinische Wochenschrift, in which von Economo described encephalitis lethargica for the first time [5]. This sleeping sickness he described, characterized by three predominant types, would soon turn into an epidemic.

epidemics, the diagnoses of EL, PEP, and influenza were all based on clinical findings [20], and confusing. Encephalopathies caused by influenza usually showed brain congestion, edema, and swelling, and not the typical polioencephalitic EL lesions [6]. A neurotropic form of the influenza virus was purely hypothetical and unproven at that time [21], and known mutations that would make the 1918 influenza virus neurotropic were absent in the reconstructed 1918 pandemic H1N1 strain [22]. Also, an administration of the reverse genetics 1918 H1N1 influenza virus to macaques found no evidence of direct neurotropism [23]. Autopsy of 1918 influenza victims did not show central nervous system (CNS) abnormalities either. However, a highly pathogenic avian H5N1 influenza virus strain was recently shown to enter the CNS in mice and induce neuroinflammation [24]. Thus, at least some strains of influenza virus might

directly cause encephalitis, as also described for a few human cases [25]. In order to retrospectively prove an infectious link to EL, stored brain tissue from acute EL cases would need to be retrieved in the form of formalin-fixed paraffin-embedded (FFPE) histological blocks from hospital archives. Biomolecules from FFPE samples provide an invaluable resource of historical material. However procedural variations, which may include pre-handling of tissue specimens, differences in the fixation process (ingredients, formalin concentration, pH, temperature and time) and post-fixation storage conditions, will significantly affect the retrievability of bio-molecules [26,27]. Additionally, although formalin fixation histologically preserves tissue architecture, the chemical nature of fixation detrimentally alters nucleic acids (NA) [28]. These induced

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molecular changes cause extracted NAs from FFPE tissues to be significantly fragmented, highly cross-linked with proteins, and chemically modified. There are, however, several factors that make FFPE specimens a suitable resource for genomic studies of ancient DNA (aDNA): the minimized oxidative and hydrolytic exposure to internal sections of fixed tissue will subsequently slow the rate of DNA decay [28]. Enzymatic decomposition by endogenous nucleases will be completely inactivated through fixation [29]. FFPE material will not be exposed to the same levels of exogenous microbial contamination, and the paraffinization process will additionally provide a physical barrier. The search for influenza virus A antigen in acute phase EL from such stored FFPE brain tissue samples retrieved in The London Hospital were negative, when tested with antisera against different influenza virus strains [30]. Influenza was also not considered to be associated with PEP as sequelae either [9]. With the more widespread use of molecular techniques and the subsequent molecular reconstruction of the 1918 “Spanish” influenza strain [31], several studies have re-explored influenza as a possible cause of EL. In one study published in 2001, reverse transcription PCR (RT-PCR) for influenza virus mRNA were negative from archived CNS tissue of EL and PEP patients [18]. Another RT-PCR investigation from 2003 also failed to detect influenza virus genes in archived brain samples from EL victims [32]. In situ RT-PCR for influenza was also negative in 7 PEP cases examined in 1995 [33]. Fundamentally, it must be noted that a negative PCR result does not exclude a pathogen, as technical limitations such as nonrefrigerated morgues and delayed formalin fixation resulting in a severe reduction of measurable RNA, as well as the formalin fixation itself, could all lead to false negatives [20]. Thus, by these molecular techniques, influenza could not be formally excluded as the cause of EL, but epidemiological data speaks against the influenza hypothesis.

5. Further viral candidates and the hypothesis of pathogen-induced autoimmunity As the reported clinical prodromal symptoms were unspecific, EL was overdiagnosed and an incubation time was not reliably documented, a vast range of viruses (or virus strains) having the capacity to cause the characteristic symptoms could be responsible. In theory, different infectious agents (or different strains of the same microbe) could also have caused different forms of EL – but concurrent epidemics with several pathogens seem unlikely. Of special interest is the epidemiologic peculiarity of EL in the way that it appeared as a large epidemic nearly 100 years ago and as sporadic cases thereafter. Whether this is due to an established immunity in large parts of today’s population due to (sub-)clinical infection with antigenetic similar, but less virulent infectious agents remains speculative. Viruses like those causing classical encephalitis (West Nile virus infection, Japanese encephalitis, St. Louis encephalitis, Venezuelan, Western and Eastern equine encephalitis, tick borne encephalitis, California encephalitis, lymphocytic choriomeningitis, Murray Valley encephalitis), have only rarely been associated with parkinsonism [9,25]. The same holds true for some members of the herpesvirus family (herpes simplex virus [HSV], varicella zoster virus [VZV], Epstein Barr virus [EBV], cytomegalovirus [CMV]), some picornaviruses (Coxsackie viruses, ECHO viruses, polio virus), measles virus, rubella virus, and human immunodeficiency virus [9,25]. In contrast, Parkinsonian sequelae of EL were almost unique. Many of these pathogens, like the arboviruses, would not fulfill epidemiological criteria. A very early candidate was HSV, which was isolated from some, but not many, fatal EL cases. However, the additional isolation from healthy individuals failed to make the link

persuasive [9]. Immunohistochemical investigations of an acute phase EL brain tissue sample and a PEP sample showed no evidence of HSV, CMV, influenza B virus, rubella, mumps, or measles virus [30]. Besides the hypothesis of EL being caused by a virus directly, EL could also have developed through an immune process triggered secondarily by a pathogen causing an infection in a different anatomical site than the brain. Such a mechanism would be compatible with the absence of viral NAs in archival brain tissues of acute EL cases. Moreover, a virus responsible for EL may not be the (direct) cause of PEP either [11]. In the latter “hit and run” hypothesis, the pathogen may cause a long-lasting immune response in the brain that persists many years after the primary infection has resolved [25]. Thus, a pathogen-induced secondary ‘epidemic’ autoimmune disease in susceptible individuals might have been the cause of PEP, even more likely than of EL. However, the strong association between PEP and EL is questioned by some authors today [11] as the diagnosis of EL and PEP was subjective and imprecise. Interestingly, poststreptococcal acute disseminated encephalomyelitis and Sydenham’s chorea resemble EL, and elevated anti-basal ganglia auto-antibodies have been demonstrated [34]. A basal ganglia autoimmune process has been postulated for classical EL [35], following a primary infection by inducing auto-antibodies directed against neuronal structures. Unfortunately, the lack of patient sera for autoantibody examination makes such studies difficult [8]. In the early 1920s, similarities between EL and poliomyelitis were seen and a common etiology was suggested as they apparently belonged to the same group of diseases [17]. In a recent study [10], electron microscopy showed structures in midbrain neurons from historical and modern EL patients, and one PEP case, which the authors called virus-like particles and which they thought were similar to enterovirus particles. Immunohistochemistry with anti-poliovirus- and anti-Coxsackie virus B-antisera demonstrated staining in neurons, neuropil, and microglia of EL brains, however, also in some control cases. A single 97 bp RNA fragment with high similarity to multiple human enteroviruses was amplified from an acute EL case using consensus primers [10]. While the authors acknowledge the molecular studies to be preliminary, the use of rigorously controlled aDNA facilities required to exclude contamination of the material from external sources of NAs and the amplification protocol is not stated. Although the isolation of viral RNA from FFPE samples is technically challenging, there are lines of thought that argue RNA stability in various post-mortem contexts. Evidence for its long-term persistence in archived material is documented [31,36].

6. Paleovirology and high-throughput sequencing pipelines HTS has revolutionized the study of degraded NAs (in particular, aDNA), providing a powerful approach for the investigation of historical diseases. The capacity of the platforms can be used to explore NAs in minute concentrations and in heterogenous NA pools. Prior to this, the field was limited to sequencing small fragments amplified through PCR or direct cloning. Despite the considerable historical and scientific interest that exists in identifying links between ancient pathogens and epidemics, few paleomicrobial studies have been of sufficient resolve or experimentally sound due to disregard of established aDNA practices [37]. Nevertheless, with careful circumspection of aDNA techniques, a broad range of pathogens (with either RNA or DNA genomes) could be screened in archived material using both specific and unbiased HTS approaches to potentially unravel important information in relation to historical diseases, such as EL (Table 1). For further information from a modern sample context, a recent review summarizes genomic

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Table 1 Assumptions and considerations for the retrieval of paleo-microbial nucleic acids from formalin-fixed paraffin-embedded (FFPE) specimens from patients that have died of acute encephalitis lethargica (EL). Assumptions 1) FFPE samples from patients who died of acute stage EL were correctly diagnosed as having EL based on accurate historical medical records and histopathological characteristics. 2) FFPE specimens from acute EL were processed in a timely manner and thus are unlikely to be contaminated by exogenous microbes prior or during the fixation process. Thus, any isolated nucleic acids in the FFPE sample are either directly associated with the pathology of the disease, constitute part of the typical human viral flora, or are from latent microorganisms (i.e., herpesviruses, JC virus). 3) By using current HTS methodologies, detectable quantities of paleo-microbial RNA and DNA in FFPE specimens from acute EL cases remain in the respective samples after fixation. Considerations 4) Ancient DNA protocols and facilities are required for molecular studies into FFPE samples from acute EL cases. 5) Removal of cross-links is a crucial step to increase the levels of retrievable pathogen nucleic acids. 6) The authenticity of isolated paleo-microbial nucleic acids must be established using strict criteria. 7) The implementation of ancient DNA practices and criteria of authentication will not guarantee that sequences are endogenous if the samples are contaminated prior to analysis and/or during the fixation process. In these circumstances, even careful use of ancient DNA criteria of authenticity will prove irrelevant, as they will be unable to verify the authenticity of microbial nucleic acid in the sample. 8) The retrieval and authentication of paleo-pathogen nucleic acids from FFPE EL samples is not proof of causation, but of the presence of microbes within that sample. 9) Novel pathogens, highly dissimilar to any pathogen sequence in GenBank, will go unidentified using current homology or similarity based searches. However, the comparison of known pathological samples to known non-pathological samples may be indicative of suspect organisms or viruses.

applications of virus identification in emerging outbreaks and the discovery of novel viruses in acute and chronic diseases [38]. Nowadays, most viral discovery studies typically employ physical virion enrichment and enzymatic pre-treatment steps prior to sequence-independent amplification and sequencing on HTS platforms. The rationale behind these methods is to reduce the high levels of background host NAs from the crude lysate and therefore, increase the sensitivity of viral detection [39]. Unfortunately, such an approach would be largely inapplicable to FFPE tissue, as the adverse effects of fixation (as well as the deparaffinization) would damage viral capsids and make viral NAs amenable for digestion via nucleases. Following the reduction of cross-links and extraction of NAs, several HTS options become available (Fig. 3). Direct shotgun sequencing provides one such approach, especially if no particular pathogen is suspected of being causally linked to EL. However, without prior viral enrichment, host NAs will inevitably represent the dominant sequenced reads. Here, the use of targeted depletion mechanisms such as endonuclease digestion [40] to selectively degrade non-viral NAs from DNA libraries may increase the sensitivity of this approach. In theory, subtractive hybridization of host-derived sequences (e.g. ribosomal DNA) may also be used [38]. However, the feasibility and effectiveness of this technique remains to be tested, as outlined by the paucity of studies employing such an approach in this field. Importantly, there are inherent benefits in performing an initial direct shotgun-sequencing attempt: the shear throughput of these platforms could simultaneously sequence associative pathogen NAs using minimal prior amplification through sequence mining and pathogen screening. Sequence reads can provide useful information including a molecular snapshot of sequence diversity and concentration within the sample material, as well as prediction of damage patterns within the sample and thereby a means to authenticate retrieved endogenous ancient sequences [41,42]. When searching for trace pathogen NAs the complexity and concentration of host derived NAs may cause problems with the sensitivity of the sequencing approach. Furthermore, the destructive nature of the fixation process will further reduce the limited quantities of paleo-microbial NAs, pushing it to the limits of detection with current molecular methods. Sequence capture methods provide both an alternative to the strength of direct shotgun sequencing or a follow-up approach once paleomicrobial sequences have been identified. Similarly, the method provides a paradigm shift away from PCR in aDNA studies due to the fragmented nature of NAs obtained from historical biological material. In this context, it provides advantages over another commonly

applied viral discovery tool, consensus PCR, which uses degenerate PCR primers designed across commonly shared regions within a viral family. Sequence capture methods present a means to enrich target sequences by pre-designing probes that are used to “fish out” desired target NAs based on prior sequence knowledge [43]. Several hybridization capture methods are available. However, all consist of a target probe immobilized either on a solid matrix or on a bead. The employment of target capture techniques require a priori sequence information for probe design that unfortunately forms part of the paradoxical argument of pre-designing molecular tools for currently un-discovered novel infectious agents. Unequivocally, the stringency of the capture approach can be relaxed to more easily capture divergent sequences to the hybridization probes. These approaches have been successfully used to fish out NAs of interest such as viruses in modern clinical samples [44], complex mixtures in FFPE samples [45], and other aDNA source material where exogenous DNA was in excess [46]. Principally, even in acute EL cases, pathogen NAs will represent a small proportion of the total endogenous NAs. Moreover, the inherent difficulties associated with capture of aDNA may cause sub-optimal performance of this method as emphasized by a number of recent studies, which showed that high amount of endogenous target molecules are crucial in reducing the likelihood of increased redundant clonal reads [45,47]. These studies emphasize the need for screening of “optimal” samples for current molecular approaches. 7. Perspective As outlined above, EL was presumably an infectious disease and most likely of viral origin. Because of its epidemic nature in temperate regions in the winter months, a respiratory or oral route of transmission is plausible. Several molecular and immunohistochemistry studies in the past could not find convincing, detailed, or reproducible traces of candidate microbes due to methodological and specimen limitations. However, with the development and rapid utilization of HTS an alternative option to thoroughly explore the nature of EL has arrived. An analysis of shotgun sequence reads will provide important clues to the taxonomic diversity present within the sample, and potential pathogen candidates for followup studies. On revealing microbes in fixed brain tissue, the clinical significance of potential pathogen candidates will need to be thoroughly assessed by revisiting adaptations to Koch’s postulates [48]. Certainly, this may prove challenging if the agent of the disease has disappeared in its entirety. Importantly, the molecular retrieval of NA sequences of various latent human herpesviruses is to be

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Fig. 3. Proposed methodology for high-throughput sequencing of extracted nucleic acids from archived formalin-fixed paraffin-embedded (FFPE) brain samples from acute cases of encephalitis lethargica (EL). Texts in white boxes refer to a specific consecutively numbered analysis step (1–7; see below). Capital letters (A–D) in small attached boxes to the respective steps indicate specific considerations (see below). Texts in gray boxes are specific questions relating to the analyzed sample. Analysis steps: 1. Archived FFPE brain tissue samples from patients who have died from acute EL to be identified. When possible, samples with documented medical records and information regarding fixation processes and storage should be chosen. Indirect measures such as electron microscopy could be used to evaluate tissue preservation and assess the level of tissue necrosis [10]. 2. Sample extraction involves the reduction of bio-molecular cross-links and use of ancient DNA facilities. 3. Whole genome amplification protocol (Phi29). Procedure dependent on quality and fragmentation of circular DNA molecules. 4. Specific sequence capture performed on suspect pathogen at species, genus or family level(s). Capture probes can be designed from known current pathogen sequences in public sequence databases such as GenBank. 5. Experimental design based on sequence output. Full genome recovery dependent on pathogen biology, divergence to known sequenced strains and sequence coverage attained. 6. Broad sequence capture performed across a large range of organisms at the genus or family level. Barcoding regions (e.g. ribosomal DNA in bacteria) can be targeted with tiling capture for species identification. 7. Metagenomics may include sequence-independent amplification and host depletion mechanisms (e.g. use of blocking oligonucleotides or direct removal of background host nucleic acids) to increase sensitivity prior to sequencing. Specific considerations: A, Molecular assessment of nucleic acids: amplifiable ancient human DNA present (confirmation via real-time PCR) and sequence length assessment (Agilent 2100 Bioanalyzer). B, Assessment of sequence reads: (i) pathogen sequence mining and identification (ii) bio-computational authentication of ancient DNA using damage pattern assessment and (iii) sequence mining for total sequenced reads (different species – human/other), under and over-represented regions (coverage and clonality). C, Clinical and epidemiological data gathered for verification of pathogen-induced symptoms – Could this pathogen cause EL? D, Examination of pathogen sequences: (i) homology and sequence divergence to known pathogens for verification of authenticity, or potential source of contamination (phylogenetics), (ii) design of real-time PCR assay for testing prevalence across multiple archived brain material from acute EL cases. Also test negative EL cases to ensure that the microbe is not prevalent in control groups.

expected in the fixed brain tissue (containing neurons, glia, blood vessels, and lymphocytes), such as HSV, VZV, EBV, CMV, human herpesviruses 6 and 7, and also JC virus. In addition to the investigation of brains from cases with acute EL by HTS, control CNS tissue from patients who have died of other conditions should be included in the analysis for comparison. Sequences of inactivated endogenous retroviruses may also be found. A potential cause of EL and candidates for a starting point using HTS because of their routes of transmission and epidemiology could be the paramyxoviruses, picornaviruses (including the enteroviruses), and adenoviruses. Future molecular studies should also aim to obtain the complete genome of the recently identified enterovirus, using the published sequence as primer, and confirm the virus’ prevalence across archived material from both EL and non-EL cases. Funding None. Competing interests None declared. Ethical approval Not required.

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